Process of combatting fungal infestations with ring chlorinated pyrimidines



United States Patent 3,227,612 PROCESS OF COMBATTING FUNGAL IN- FESTATIONS WITH RING CHLORIWATED PYR'P/HDINES Herman Gershon, North Bergen, NJ., assignor to Pfister Chemical Works, Inc., Ridgeiield, N.J., a corporation of New Jersey No Drawing. Filed July 23, 1962, Ser. No. 211,365 11 Claims. (Cl. 16739) This invention relates to methods for combatting or controlling fungi. Among the industrial areas where infestations of fungi require control can be mentioned the pulp, paper and textile industries, where organisms such as Penicillium spp., Aspergillus niger, T richoa'erma viride, and Myrothecium verrucaria are encountered. Certain of these organisms digest cellulose readily, and, unless controlled, large economic losses result. For example, during prolonged storage of textiles in warehouses, or in tropical areas, or during transportation while in the holds of ships, where relatively high humidity exists, fungal attack is a major problem. iarticularly important are protective agents which alford satisfactory control of the fungal infection and are easily removed when desired.

Of importance also are substances whose vapor are antifungal and especially sporicidal. Such substances are useful in elfecting sterilization particularly where it is necessary to efiect the sterilization at relatively low temperatures, i.e., the so-called Cold Sterilization, to

avoid injury to the articles subjected to sterilization.

Ethylene oxide has been suggested for this purpose but the use of ethylene oxide is objectionable for a number of reasons among which may be mentioned, (1) ethylene oxide is flammable when its vapors are combined with air, in fact at concentrations of about 3% ethylene oxide, explosive mixtures result, (2) ethylene oxide is a gas at room temperature and hence special equipment must be used to confine it, (3) ethylene oxide loses its sterilization or anti-fungal properties under humid conditions, and (4) ethylene oxide does not adequately control or attack microbal spores.

It is a principal object of the present invention to provide a method of controlling or combatting fungi which can be used, for example, in destroying or preventing the growth of fungi including spore formation and is simple and eifective to practice.

Another object of this invention is to provide such method which can be used to elfect sterilization of objects at ambient or relatively low temperatures, below the boiling point of Water, effective under all conditions of'humidity encountered, and this without creating fire or explosion hazards, and without requiring special equipment.

Still another object of this invention is to provide a method of protecting material subject to fungal growths, during storage, or shipment in containers, which method involve the simple addition or" anti-fungal material to the container to provide the desired protection and this without injury to the contents of the container.

A further object of this invention is to provide a method of protecting cellulosic and other material against fungi, employing anti-fungal compositions which can readily be removed from the protected material and this without injury to the protected material and without leaving objectionable odor.

Other objects and advantages of the present invention will be apparent from the following description thereof.

In accordance with this invention the cellulosic or other material to be protected is exposed to the vapors or impregnated with a solution of a ring chlorinated pyrimidine having the formula:

s u C in which R H, Cl, CH C H or CH CI; R =H, Cl, CH and R =H, Cl, CH C H or CH Br, and when R is hydrogen at least one of R and R is chlorine.

Examples of these compounds found effective as antifungal agents and whose vapors are anti-fungal and show exceptional sporicidal properties are given in Table I The numbering of the positions of the substituents in the pyrimidine nucleus is as follows:

all ('15 These pyrimidines can be prepared by any of the known procedures of producing such compounds. The dichloroand trichloropyrimidines can be prepared from the uracils and 5-chlorouracils, respectively, by reaction with phosphorus oxychloride. The 6-substituted pyrimidines can be prepared by condensing the appropriate B-ketoester with thiourea in the presence of sodium ethylate to 6-substituted-2-thiouracil, according to the general procedure of Anderson, et a1. (G. W. Anderson, I.-F. Halvarstadt, W.'H. Miller and R. O. Roblin, Jr., J. Am. Chem. Soc. 67, 2197 (1945). The conversion of the thiouracils is accomplished by hydrolysis with chloroacetic acid and .hydrochloricacids. The uracils are chlorinated in the S-positions with sulfuryl chloride by the method of Barrett, Goodman, and Dittmer (LAm. Chem. Soc. 70, 1753 (19.48). Theprocedure involving condensing acetamidewith ethylchloromalonate in the presence of shodium ethylate to yield 5-chloro-2-methylpyrimidine- 4,6-diol and reacting this diol with phosphorus oxychloride to produce 2-methyl-4,5,6-trichloropyrimidine, can be chloromethylpyrimidine; and 2,4,5,-tetrachloropyrimie dine are preferred because they are outstandingly efficient antifungal agents and their vapors are exceptionally effective sporicidal agents.

The pyrimidine derivatives can be applied to the material to be protected in solution form. For example, fabrics may be dipped into a solution of the compound in a solvent followed by evaporation of the solvent, or the solution can be sprayed onto the fabric. Solutions having a concentration of about 0.1% by weight or higher (these compounds are miscible with the solvents of choice in all proportions) can be used. Acetone, the lower alcohols such as methanol, ethanol, the propanols, carbon tetrachloride or perchlorethylene, are suitable solvents. A 0.5% to 1% solution of compound in acetone gives satisfactory protection to the cellulosic fabrics against attack by fungi which develop in humid atmospheres.

For protection against fungal spores or fungus growths in cellulosic or other materials in storage or transportation a small amount of the pyrimidine derivative, preferably one solid at ambient temperatures, is introduced into the container or hold for the materials. Desirably from 1.5 to 33 milligrams per 1000 ml. of space, preferably from to 20 milligrams per 1000 ml. of space is employed. Thus, for example, shipment of cellulosic material such as fabrics in containers in which a small amount of pyrimidine is introduced results in effective protection; the vapors result in effective control of fungi and prevent germination of spores.

For sterilization purposes the items are subjected to vapors of the pyrimidine. These vapors are not flammable, do not produce explosive mixtures with air, are relatively non-toxic, are not deleteriously affected by humid atmospheres and are either liquid or solid, most are solid at ambient temperatures, so that no special equip ment required for introducing and confining a gaseous medium such as ethylene oxide need be used.

The compounds herein disclosed can be easily removed from the fabric by treatment with an organic solvent such as perchlorethylene.

The following tests demonstrate the effectiveness of the pyrimidine compounds in protecting cotton against fungi. This anti-fungal test is that prescribed by The American Society for Testing Materials, Committee D-13, ASTM Standard on Test Materials, 77, Philadelphia, 1943. Six inch by one inch strips of white cotton cloth weighing 576580 mg. each were used. These strips were immersed in a 0.2% solution of 2-chloromethyl-4,5,6-trichloropyrimidine in acetone and the acetone was allowed to evaporate. Each strip contained from 10 mg. to 12 mg. of the protective agent, i.e., about 0.2%.

French square bottles (i.e., bottles square shaped in cross-section) of 16 02. capacity were used as the incubation chambers. Into each bottle was placed a standard liquid inorganic nutrient medium for fungi having the following inorganic salt composition:

Inorganic salts: gms./liter K HPO 1.3940 MgSO 0.7395 NH NO 1.0006 NaCl 0.005 Fe, Zn and Mn or $0.; 0.001

Into each bottle, while on its side, was placed a glass filter sheet to act as a support for the cotton strip and to prevent its submersion in the solution. Sufiicient inorganic salt solution was introduced into each bottle to soak the glass filter sheet therein, and the cotton strip was placed on the filter sheet. The cotton strip became soaked with inorganic medium by capillarity, and the bottles were each capped with Pyrex glass wool and autoclaved for 20 min. at p.s.i.g. To each sterile chamber, thus produced, was added 1 ml. of spore suspension containing 6 10 spores/ml. Penicillium sp. and M. verrucaria were used as the test organisms. Controls for each 3. organism consisted of one chamber containing unprotected cotton cloth, one chamber containing no cotton cloth, and one chamber which remained uninoculated.

To test the vapor effect of the compound, a bottle was prepared with unprotected cloth into which was also inserted a 1 ml. beaker containing 2 or 3 mg. of crystals of test compound. Three replicates of each bottle were used.

The bottles, prepared as above described, were incubated at 28-30 C. for 7-10 days. At the end of this period, it was observed: the uninoculated bottles and the bottles containing no cotton showed no fungal growth, the bottles with unprotected cotton showed good fungal growth, and the bottles containing cotton cloth impregnated with 2-chlorornethyl-4,5,6-trichloropyrimidine showed no fungal growth as did the bottles containing unpreimpregnated cotton which was exposed to the vapors of 2-chloromethyl-4,5 ,S-trichloro pyrimidine.

This test demonstrated the effectivenes of 2-chloromethyl-4,5,6-trichloropyrimidine in protecting cotton and particularly cotton cloth against infestation by the fungi employed as test organisms.

Table II presents the anti-gungal data on the ring chlorinated pyrimidines as determined by the disc-plate method. The organisms employed in this test included five fungi (A. niger, T. viride, M. verrucaria, Penicillium sp., and T. mentagrophytes). The media employed were as follows: for all organisms Sabourauds dextrose agar was employed, except 10% beef serum was added to the medium for T. mentagrophytes.

The anti-fungal tests were conducted in the following manner: a spore suspension of the respective organisms was prepared in 10 ml. of terile 0.9% NaCl solution from a 7 day culture of each fungus on a test tube plant of Sabourauds dextrose agar at 2830 C. The 10 ml. of spore suspension was then incorporated into 50 ml. of medium at 45 C., and 15 ml. portions of inoculated medium were poured into 10 cm. Petri dishes and allowed to harden. Filter paper discs, 12 mm. in diameter, which had previously been impregnated with 3 levels of compound, 10*, 10 and 10 micrograms/disc, were placed on the hardened agar and allowed to incubate at 2830 C. for 5 days. The lowest level of compound causing inhibition was recorded.

In Table II, A.N. is Aspergillus niger; T.M. is T rich0- phyton mentagrophytes; P. sp. is Penicillium sp.; M.V. is Myrothecium verrucaria; and T.V. is T richoderma viride.

TABLE II [Anti-fungal data] Micrograms of compound/ disc causing inhibition Pyrimidine A.N. T.M. P. sp. M.V. T.V.

2,4-dich1oro- 10 10 10 10 10 2,4-dich1oro'G-methy1- 10* 10 10 10 2,4dichloro-6-ethyl- 10 10 10 10 10 2,4-dichloro-5methyl- 10 10 10 10 10 2-rnetl1ylt,5,6-trichloro 10 10 10 10 10 2-ethyl-4,5,6-trich1oro- 10 10 10 10 10 G-methyl-2,4,5-trichloro- 10 10 10 10 10 6-ethyl-2,4,5-trichloro- 10 10 1t) 10 10 5-methyl-2,4,6-trichloro- 1 10 1t) 10 10 10 2-chlorornethyl4,5,6-

trichloro- 1O 10 10 10 10 6-br0mornethyl-2,

trichloro- 10 10 10 10 10 Table III presents the results of the tests employing the vapors of these ring chlorinated pyrimidines on the spores of 3 fungi (A. niger-A.N., Penicillium sp.-P. sp., and T. viride-T.V.).

The medium employed was Sabourauds dextrose agar, and the tests were conducted in the following manner: 1 ml. of a spore suspension of the respective organism containing 6 10 spores in 0.9% NaCl was added to ml.

of medium at 45 C. Three to five ml. of each inoculated medium was added per chamber to each of 3 chambers in sterile quadriplate Petri dishes. To the fourth chamber was added 26 mg. of solid compound or 1 drop of liquid compound on a 12 mm. sterile filter paper disc. The dishes were incubated at 2830 C. for 5 days. A 5 mm. portion of agar was cut from all portions of medium which showed no apparent fungal growth and was subcui'tured in ml. of Sabourauds liquid medium (Difco) enriched with 0.1% yeast extract (Difco). Incubation at 28-30 C. was carried out for 2 weeks. If no fungal growth appeared on subculture, the compound was considered sporicidal, but if there was fungal growth on subculture, the compound was considered soristatic.

In table III, C indicates sporicidal, S indicates sporistatic, indicates no fungal inhibition, and a number indicates the quantitative percent of the area showing no fungal growth. This table gives the results of the same tests employing the same test conditions on the same spores employing the known anti-fungal agents identified in the table.

TABLE III Efiecf of vapors of ring chlorinated pyrimidines on fungal spores Pyrimidine sp.

OOOOOOOOOOOOOOOO SOOOOQOOOOOOOOOO OOOOOOOOOOOOOOOO COMPARATIVE TEST RESULTS C ompound:

S-quinolinol 5-chloro-8-quinolinol 5-iod0-8-quinolinoL mercurous ch1ori(le mercuric chloride mere phenyl mercuric acetate c.++++aa The superiority of the ring chlorinated pyrimidines of this invention as anti-fungal agents for controlling spore germination is at once evident from the above table. It will be noted that all of the identified ring chlorinated pyrimidines are completely effective under the test conditions to kill the spore; the letter C in the above table indicates 100% control, i.e., no fungal growth under the test conditions.

It will be noted that the present invention provides an efiicient process for the control of fungi, particularly, those which infest cellulosic materials, such, for example, as cotton fabrics, paper and pulp. The ring chlorinated pyrimidines can be applied to materials to be protected by impregnating same with solutions thereof to give temporar protection, for example, during storage or shipment and can thereafter be readily removed by rinsing the treated material with an organic solvent such as methanol, ethanol, isopropanol, acetone, carbon tetrachloride, trichlorethylene, or perchlorethylene.

Prolonged protection is obtained by enclosing a relatively small amount of the identified ring chlorinated pyrimidine in the sealed package or container for the cellulosic or other material to be protected. The vapors provide an atmosphere within the package or container which destroys fungi and prevents spore germination.

Cold Sterilization can be effected by exposing the items to be sterilized to the vapors of the identified ring chlorinated pyrimidines.

It will be understood that this invention is not to be limited to the disclosure herein except a indicated by the appended claims.

What is claimed is:

1. The process of cobatting fungal infestations in material subject to infestation by fungi and preventing spore germination in said material, which process comprises contacting said materials with the vapors of a ring chlorinated pyrimidine from the group consisting of 2,4- dichloro-, 4,5-dichloro-, 2,4,5-trichloro-, 2-,4,6-trichloro-, 4,5,6-trichloro- 2,4,5,6-tetrachloro-, 2,4-dichloro-6-methyl-, 2,4-dichloro-6-ethyl-, 2,4-dichloro-5-m'ethyl-, Z-methyl- 4,5,6-trichloro-, 2-ethyl-4,5,6-trichloro-, 6-methyl-2,4,5- trichloro-, 6-ethyl-2,4,5-trichloro-, 5-methyl-2,4,6-trichloro-, 2-chloromethyl-4,5,6-trichloro-, and 6-bromomethyl-2,4,5-trichloro-pyrimidines.

2. The process of protecting against fungal growth and germination of spore the contents of a container which contents are subject to fungal infestation, which process comprises introducing into said container 1.5 to 33 milligrams per 1000 ml. of capacity of said container of a ring chlorinated pyrimidine from the group consisting of 2,4-dichloro-, 4,5-dichloro-, 2,4,5-trichloro-, 2,4,6-trichloro-, 4,5,6-trichloro-, 2,4,5,6-tetrachloro-, 2,4-dichloro-6-methyl, 2,4-dichloro-6-ethyl-, 2,4-dichloro-5-methyl-, 2-n1ethyl- 4,5,6-trichloro-, 2-ethyl-4,5,6-trichloro-, 6-methyl-2,4,5-trichloro-, 6-ethyl-2,4,5-trichloro-, 5-methyl-2,4,6-trichl0r0, 2-chloromethyl-4,5,6-trichloro-, and 6-bromoethyl-2,4,5- trichloro-pyrimidines.

3. The process of protecting against fungal growth the contents of a sealed container which comprises introducing into said container from 10 to 20 milligrams of 2,4,S-trichloropyrimidine in the container for each 1000 ml. of space in said container to provide within said sealed container an atmosphere containing the vapors of said pyrimidine.

4. The process of protecting against fungal growth the contents of a sealed container which comprises introducing into said container from 10 to 20 milligrams of 4,5,6-trichloropyrimine in the container for each 1000 ml. of space in said container to provide within said sealed container an atmosphere containing the vapors of said pyrimidine.

5. The process of protecting against fungal growth the contents of a sealed container which comprises introducing into said container from 10 to 20 milligrams of 2,4,5,6-tetrachloropyrimidine in the container for each 1000 ml. of space in said container to provide within said sealed container an atmosphere containing the vapors of said pyrimidine.

6. The process of protecting against fungal growth the contents of a sealed container which comprises introducing into said container from 10 to 20 milligrams of 4,5,6-trichloro-2-ch1oromethylpyrirnidine in the container for each 1000 ml. of space in said container to provide within said sealed container an atmosphere containing the vapors of said pyrimidine.

7. A closed container the contents of which are subject to fungal infestation and spore germination having said contents protected by being surrounded by the vapors of a ring chlorinated pyrimidine from the group consisting of 2,4-dichloro-, 4,5-dichloro-, 2,4,5-trichloro-, 2,4,6-trichloro-, 4,5,6-trichloro-, 2,4,5,6-tetrachloro-, 2,4-dichloro-6-methyl-, 2,4-dichloro-6-ethyl-, 2,4-dichloro-S-methyl-, 2-methyl-4,5,6-trichloro-, 2-ethyl-4,S,6-trichloro-, 6-methyl-2,4,5-trichloro-, 6-ethyl-2,4,S-trichloro-, 5-methyl-2,4,6-trichloro-, 2-chloromethyl-4,5,6-trichloro-, and 6-bromornethyl-2,4,S-trichloro-pyrimidines.

8. A closed container the contents of which are subject to fungal infestation and which contents are protected by being surrounded by the vapors of 2,4,5-trichloro-pyrimidine.

9. A closed container the contents of which are subject to fungal infestation and which contents are protected by being surrounded by the vapors of 4,5,6-trichloro-pyrimidine.

10. A closed container the contents of which are subject to fungal infestation and which contents are protected by being surrounded by the vapors of 2,4,5,6-tetrachloro-pyrimidine.

11. A closed container the contents of which are subject to fungal infestation and which contents are protected by being surrounded by the vapors of 4,5,6-trich1oro-2- chlorornethylpyrimidine.

References Cited by the Examiner UNITED STATES PATENTS 2,649,397 8/1953 Ballard 16733 0 0 2,812,328 11/ 1957 Hepworth 16733 2,940,895 6/1960 Overbeek 167-33 FOREIGN PATENTS 5 793,749 4/1958 Great Britain.

685,103 12/1952 Great Britain.

OTHER REFERENCES Gershon et a1.: J. of Organic Chemistry, vol. 26, June 1961, pp. 1874-1876.

Smith et a1.: 1. of Organic Chemistry, vol. 20, July 1955, pp. 829-38.

15 LEWIS GOTTS, Primary Examiner. 

1. THE PROCESS OF COBATTING FUNGAL INFESTATIONS IN MATERIAL SUBJECT TO INFESTATION BY FUNGI AND PREVENTING SPORE GERMINATION IN SAID MATERIAL, WHICH PROCESS COMPRISES CONTACTING SAID MATERIALS WITH THE VAPORS OF A RING CHLORINATED PYRIMIDINE FROM THE GROUP CONSISTING OF 2,4DICHLORO-, 4,5-DICHLORO-, 2,4,5-TRICHLORO-, 2,4,6-TRICHLORO, 4,5,6-TRICHLORO- 2,4,5,6-TETRACHLORO-, 2,4-DICHLORO-6-METHYL-,2,4-DICHLORO-6-ETHYL-, 2,4-DICHLORO-5-METHYL-, 2-METHYL4,5,6-TRICHLORO-, 2-ETHYL-4,5,6-TRICHLORO-, 6-METHYL-2,4,5TRICHLORO-, 6-ETHYL-2,4,5-TRICHLORO-, 5-METHYL-2,4,6-TRICHLORO-, 2-CHLOROMETHYL-4,5,6-TRICHLORO-, AND 6-BROMOMETHYL-2,4,6-TRICHLORO-PYRIMIDINES. 